Lower Limb Ground Reaction Force and Center of Pressure Asymmetry During Bodyweight Squats

Background Performance asymmetries between the lower limbs have been reported across a variety of variables and for numerous motor tasks including double leg squats. Additionally, the degree of symmetry is often used as a recovery metric during rehabilitation programs. Hypothesis/Purpose The purpose of this investigation was to examine leg asymmetry during a bodyweight double leg squat task and assess the effects of squat speed in a physically active population. Study Design Cross-over Study Design. Methods Eighteen healthy individuals completed two sets of 20 squats at two tempos (preferred tempo and 60 bpm) while ground reaction force and center of pressure data were recorded using dual force plates. Peak vertical ground reaction force, force impulse, and center of pressure (COP) standard deviation in the anterior-posterior (AP) and mediolateral (ML) direction were calculated and analyzed to identify any differences between legs, tempo, and as a function of repetitions. Significance was set at ρ ≤ .05. Results The subjects exhibited greater ground reaction forces during the self-paced tempo compared to the metronome-paced tempo (F1,79 = 14.48, p < .001) with the preferred leg generating larger values than the non-preferred leg during the self-paced condition. There was also a significant tempo x leg interaction for force impulse (F1,79 = 5.927, p = 0.015). A greater amount of COP variability was found in the preferred leg compared to the non-preferred leg in both the AP (F1,79 = 30.147, p < 0.001) and ML (F1,79 = 41.204, p < 0.001) directions. Conclusions These findings highlight the importance of considering multiple levels of analysis when assessing lower limb symmetry as separate variables may provide differential evidence for asymmetry. Practically, these results emphasize the need for coaches and practitioners to consider different degrees of lower limb asymmetries that may impact the development and design of strength and rehabilitation programs. Level of Evidence 3

INTRODUCTION
The double leg squat, in either the loaded or bodyweight form, serves as a common functional exercise used in weight training and rehabilitation settings. Successful execution of the movement requires the coordination and control of numerous muscles, as well as the maintenance of postural stability. Given the dynamic nature and functional demands of the double leg squat, it is often assumed that symmetric control exists between the right and left legs, as evidenced in part by the common approach to only collect unilateral measurements. 1,2 Furthermore, it is often postulated that symmetrical kinematics derive from symmetrical force production and symmetrical joint moments during a squat movement. 3 However, redundancies present in the neuromuscular system afford the possibility of bilateral asymmetries across different levels of analysis. Thus, caution should be exercised when interpreting the existence of bilateral symmetry, particularly when used to determine exercise prescription on the common assumption that the demand will be equally split between the two extremities.
Bilateral asymmetries have been examined across a variety of motor tasks including countermovement jumps, single leg countermovement jumps and hops, back squat, gait, and upright standing. Similar to evidence found in gait, 4 conflicting findings exist on whether bilateral differences are present during the squat movement, and this is evident across multiple levels of analysis (e.g., kineticjoint torque, ground reaction forces or kinematic measurements). However, few studies have addressed the dynamic balance component (e.g., postural sway) of the squatting movement. Flanagan and Salem 5 used a modified center of pressure (COP) variable that was derived as the distance from the ground reaction force (GRF) application point to the ankle joint and showed significantly greater distances for the left lower limb as compared to the right limb across various loading conditions. Using more typical COP measurements, Kohn and colleagues 6 reported postural stability measurements as a function of upward gaze direction during squatting and found that a downward, compared to an upward gaze, reduced COP displacements. However, the preceding evidence focuses on a whole-body COP metric and have yet to address the question of whether the maintenance of balance during this dynamic movement is achieved equally from the right and left limbs.
Although limb asymmetry is commonly interpreted as indicative of pathology, evidence from gait analyses suggests that even healthy individuals display asymmetrical behavior. 4 In fact, most individuals exhibit a degree of lower limb performance preferences manifested as a strong functional advantage for one limb in asymmetrical tasks. 7 Specifically, staggered and tandem standing resulted in increased asymmetrical weight distribution between the legs as compared to a side-by-side posture. 8 During locomotion, an asymmetrical second peak of the vertical GRF curve was found and influenced by walking speed while other kinetic properties remained symmetrical. 9 Collectively, it is important to understand the contributing factors to limb symmetry and to explore how the body adapts to changes in environmental or tasks constraints that may lead to functional advantages for one of the lower limbs.
Bilateral asymmetries can be calculated using a variety of equations that typically revolve around distinctions like dominant versus nondominant, right versus left, stronger versus weaker, or preferred versus nonpreferred limbs. Additionally, different metrics (e.g., symmetry indices, statistical analysis) have been used to examine the degree of symmetry between the right and left legs. In healthy gait, some investigations analyzed ground reaction forces while others examined spatial-temporal properties of walking and running. 4 Additionally, postural control asymmetries have typically been addressed using center of pressure (COP) with evidence of asymmetry found in both static and dynamic balance tasks. 8,10,11 Examination of bilateral asymmetries over multiple levels of analysis may provide insight into possible underlying mechanisms that facilitate successful task execution and has the potential to guide practitioners in designing effective training and rehabilitation exercises.
The current investigation explored whether leg asymmetry exists during a bodyweight double leg squat task and assessed the effects of squat speed in a physically active population. First, we examined whether typically used kinetic variables (e.g., vertical ground reaction force -GRF v ) demonstrated bilateral asymmetries during the double leg squat. 12 A secondary aim addressed the lack of evidence related to postural stability asymmetries by quantifying center-of-pressure (COP) sway of each leg. Increased COP displacements are interpreted as an indication of less postural control and poorer balance performance. Based on the findings of Flanagan and Salem, 5 it was predicted that the left leg would exhibit greater COP displacements. Finally, the pace of movement has been shown to influence movement characteristics, 9 and we sought to examine whether this pattern of results remained consistent for self-paced and metronome-paced movements during 20 repetitions of the double leg squat task.

METHODS
Eighteen physically active young adults (10 F, 23.17 ± 1.72 years, 173.5 ± 10.18 cm, 74.18 ± 16.31 kg) were recruited from a university campus and provided informed consent prior to participation. Participants were considered active if they reported participating in physical activity a minimum of three days per week. Lower limb preference was de-termined by asking participants which leg they would use to kick a ball for maximum distance (right-preferred =12, left-preferred = 6). Participants reported no lower extremity injury that required surgery in the past year. The following protocol was approved by the Texas Christian University institutional review board prior to implementation and informed consent was obtained from all participants prior to data collection.
Participants completed two sets of 20 repetitions of double leg squats with one set performed at a preferred tempo (self-selected by each participant) while the other set was at a non-preferred tempo (metronome -60 bpm; one second descent, one second ascent). Feet were positioned shoulder width apart and arms were extended overhead during the bodyweight squats. The order of squat tempo was randomized for each subject. During all squats, participants executed the movement to a depth that was comfortable for them and a researcher observed performance to ensure that participants maintained a similar depth for the set of 20 repetitions. During the repetitive squat movement, bilateral GRF v were collected with dual force plates (AMTI, Watertown, MA, USA) using a sampling rate of 100 Hz.
Kinetic data were filtered using a Butterworth filter and a 10 Hz cut-off frequency. A researcher manually identified each squat cycle in Visual 3D (C-Motion, Inc., Germantown, MD, USA) with initiation of right knee flexion indicating squat start and termination of right knee extension indicating squat end. The squat phases were then defined as the time from initiation of right knee flexion to maximum knee flexion (descent phase) and maximum knee flexion to termination of right knee extension (ascent phase). The GRF v peak and impulse were determined for the right and left legs for the ascent phase of each squat repetition since the peak GRF typically occurs during this phase. 12 The variability of center of pressure trajectories in the anterior-posterior (AP) and medial-lateral (ML) directions for the preferred and non-preferred legs were computed by standard deviation for the entire duration of each trial.
The dependent variables (movement time, peak GRF v , F impulse , COP standard deviation -AP, COP standard deviation -ML) were analyzed in separate 2 (leg: preferred vs. non-preferred) x 2 (tempo) x 20 (repetition) repeated measures analysis of variance. Based on the sample size (n = 18), an alpha level set at p < 0.05, and an effect size set to 0.15, the current study achieved a level of statistical power equal to 0.84. Data are reported as mean ± standard deviation. All tests were performed using SPSS statistical software (IBM, Chicago, IL).

RESULTS
Mean movement time was evaluated between the selfpaced and metronome conditions through a one-way ANOVA and revealed a significant difference for movement time (F 1,37 = 20.93, p < 0.001) with longer squat times for the self-paced (1.92 ± 0.27 s; 62.5 +1.85 bpm) compared to the metronome (1.82 ± 0.13 s; 65.93 ± 3.84 bpm) condition.
The analysis of peak GRF v revealed a significant tempo x leg interaction (F 1,79 = 7.593, p = 0.006). Although individu-als executed the squat movement with a greater peak GRF v during the self-paced (Preferred: 550.27 ± 172.15 N; Non-Preferred: 518.40 ± 134.19 N) than in the metronome (Preferred: 498.96 ± 115.93 N; Non-Preferred: 508.35 ± 130.08 N) condition for both legs (F 1,79 = 14.48, p < 0.001), there were significantly larger peak GRF v values for the preferred limb compared to the non-preferred leg during the selfpaced condition. No leg difference, repetition effect, or other interactions were found for peak GRF v (Figure 1, top  panel).
There was also a significant tempo x leg interaction for F impulse (F 1,79 = 5.927, p = 0.015), with preferred and nonpreferred limbs performing differently depending upon the tempo condition. Post-hoc analysis revealed similar impulses for the preferred and non-preferred limbs during metronome condition ( Figure 2). In the AP direction, the preferred limb (3.21 ± 0.57 cm) showed greater variability than the non-preferred limb (1.92 ± 0.06 cm) with the same pattern of results observed in the ML direction (preferred: 1.50 ± 0.38 cm; non-preferred: 0.62 ± 0.3 cm).

DISCUSSION
The current study evaluated the degree of (a)symmetry in a double leg squat as a function of squat tempo and repetition. The results revealed bilateral asymmetry for peak GRF v and F impulse dependent on squat tempo. Higher peak GRF v values were observed during self-paced squats compared to metronome-paced squats in both limbs. The preferred limb exhibited a greater difference in peak GRF v across tempo conditions, while the non-preferred showed similar values across tempos. Complimenting the asymmetrical kinetic GRF variables, the results also showed that the preferred limb exhibited greater COP variability in both AP and ML directions compared to the nonpreferred limb. These findings highlight the importance of evaluating the degree of limb symmetry across multiple levels of analysis and indicate a functional specialization exists within the lower extremities when considering force production and postural control properties.
At the GRF level of analysis, the between limb peak GRF v asymmetry finding during the squat task supports some previous evidence, 13 while contrasting other results. 14 During weighted back squats performed by college athletes Newton et al. 13 reported significant bilateral differences in peak GRF v during the concentric phase of the movement with the dominant leg producing a greater peak force. However, when categorized by right and left leg, no significant

Figure 1. Significant Tempo x Leg Interactions for Peak GRF v and F impulse .
Top Panel: Peak GRF v was significantly greater for the preferred limb compared to the non-preferred limb in the self-paced condition compared to the metronome condition during bodyweight squats (p < 0.001). No other main effects or interactions were significant for peak GRF v . Bottom Panel: F impulse was significantly larger for the non-preferred limb during the self-paced condition. GRF v : vertical ground reaction force; N: Newtons. difference in peak GRF was found between limbs, seemingly indicating limb symmetry. 13 In a drop landing task, Schot, Bates, & Dufek 14 qualitatively observed bilateral asymmetries in GRF peaks at the individual level but reported no significant limb differences when compared at the group level. In alignment with Newton and colleagues, 13 the current study only considered the concentric phase of the squat. Consistent with their findings in a weighted back squat, the current results revealed that peak force asymmetries exist during bodyweight squat. However, Newton et al. categorized the dominant limb based on isokinetic strength testing and linked such strength differences to other functional asymmetries found in explosive movements. Here, preferred limb was identified using a footedness questionnaire which may exhibit differing characteristics than the asymmetric performance advantage (e.g., limb dominance, strength imbalances) observed previously. Future investigation is needed to better understand the connections between limb dominance and preference during the squat movement given that previous evidence has highlighted that injury and fatigue appear to induce greater degrees of lower limb asymmetries. 3 The functional demands of the squat movement would appear to require symmetrical kinetic properties pertaining to force production, suggesting a lack of functional specialization between the lower limbs in healthy individuals. Bilateral asymmetries have been explored in a variety of motor tasks with the collective evidence failing to provide clear insight on the generality of interlimb performance characteristics. For example, an investigation of limb dominance, foot orientation, and functional asymmetry during normal gait resulted in significant differences between the dominant and non-dominant limb for medial and lateral peak GRF and impulses. 15 In upright standing, some findings indicate that the dominant limb exhibits a performance advantage (e.g., less postural sway) over the non-dominant limb, whereas other findings typically show symmetrical postural sway between the limbs. Furthermore, motor tasks that demand asymmetrical roles (i.e., kicking, stepping on a box) between the limbs do appear to support the notion of asymmetrical control. The results of the current study suggest that the force production characteristics and postural control properties of the lower limbs in healthy individuals both reflect asymmetry but to varying degrees dependent on the tempo of the squat movement. For example, the contrasting findings between the two kinetic variables (GRF peak and impulse) suggest that further delineation may be needed between the control properties of the preferred and non-preferred limbs. A clear performance advantage for the preferred limb was observed in terms of producing peak force values during the self-paced condition of the squat movement. This advantage appears to be complemented by efficient force production in that lower impulses were produced compared to the non-preferred limb. Overall, these findings are consistent with the notion of functional specialization of the lower extremities in most motor tasks. 16 The variation of movement tempo has been used frequently during walking and running tasks to understand potential shifts of lower limb symmetries. 17,18 However, manipulation of tempo has received minimal consideration during bodyweight squats despite the relevance to reha-

Figure 2. Comparison of COP Variability in the AP and ML Directions.
Significantly greater variability was found in the preferred limb compared to the non-preferred limb in both the AP (p < 0.001) and ML direction (p < 0.001). SD-COP: standard deviation -center of pressure; cm: centimeters; AP: anterior-posterior; ML: mediolateral. bilitation approaches. Here, both the preferred and nonpreferred limbs exhibited greater peak GRF v during selfpaced bodyweight squats compared to squats executed in the metronome condition that was likely associated with an increased movement velocity. 19 This pattern of results held true for the non-preferred limb when considering GRF impulse but the preferred limb exhibited lower GRF impulse during self-paced squats compared to the metronome condition. Understanding how tempo affects force production and variability, specifically during bodyweight squats, may have an impact on therapeutic exercise prescription as rehabilitation exercises are often performed at a self-selected pace. Here, smaller degrees of asymmetry were observed in the metronome condition compared to the self-paced condition. Promoting symmetrical characteristics is often a primary goal of rehabilitation for most lower limb injuries, and the use of a metronome may facilitate an individual's ability to control the level of force production between the injured and non-injured limb.
The dynamic nature of bodyweight squats challenges the stabilization control of each leg, and the current findings revealed different postural solutions between the preferred and non-preferred leg not typically found in simple balance tasks. 20-22 The reduced COP variability observed for the non-preferred as compared to preferred leg suggests an enhanced stabilization component for the non-preferred leg during the propulsion phase of this dynamic movement. Therefore, it appears that the non-preferred leg more effectively countered the mechanical perturbations of body movements, and this was consistent for both AP and ML directions. However, future investigations should consider not only pedal preferences 8 but also training histories 16 when interpreting bilateral asymmetries.
Another aim of the current investigation was to examine the potential interaction between two types of asymmetry (ground reaction forces and COP under each foot), which has received minimal study in the literature. Significant differences were observed between the lower limbs for the GRF properties, and the non-preferred limb showed significantly lower COP displacements than the preferred limb. Previous evidence has shown the dependent nature of weight distribution on COP displacements during upright standing. For example, Rougier 22 showed that the more loaded limb exhibited larger displacements during unstable conditions, suggesting a compensatory strategy between the lower limbs in order to minimize whole body sway. Similarly, King, Wang and Newell 8 revealed asymmetries that were dependent on weight distribution across different postural configurations (staggered and tandem stances). In the current study, the less stable preferred limb showed increased displacements in both the AP and ML directions. Thus, the reduced COP motion under the non-preferred limb may aid in limiting whole-body COP displacements in a manner that achieves postural stability in both directions during squat execution. Future examinations of the relative contribution of each limb (i.e., weight distribution) to whole-body COP movements would provide insight into the ability of the postural control system to accommodate such variable asymmetries.
While the current study did not directly examine the role of fatigue, it is important to consider the relative impact of fatigue on bilateral asymmetries. A lack of a significant repetition effect suggests that fatigue did not influence the measured bilateral asymmetries (peak GRF v , COP variability, or impulse -F impulse ) during the bodyweight squat task within a healthy, active population. Interestingly, during five sets of eight repetitions of a weighted squat exercise, bilateral GRF v became more symmetric after participants were fatigued within one set, but this symmetrical behavior did not carry over between sets. 12 Similar results of increased symmetry of load distribution and knee and hip joint loading patterns were observed within a patient population (anterior cruciate ligament reconstruction) during 10 consecutive bodyweight squats completed after a fatiguing protocol. 3 The physical activity level of the participants may have mitigated the role of fatigue across the 20 squat cycles. Additionally, in contrast to typical fatigue protocols, the volume of workload used in the current study may have only resulted in a minor degree of fatigue, and future investigations should examine the process of fatigue onset with increased movement repetitions.
A few limitations of the current study need to be considered. First, the experimental instructions did not impose constraints on participants' selection of the self-paced tempo to be faster or slower than the metronome-paced tempo, and future investigations should consider individualizing the metronome tempo by manipulating a self-paced tempo by a percentage value. Second, it may be important for future investigations to consider sport-specific training that may influence bilateral asymmetries. Lastly, pedal asymmetry developed through the bilateral or unilateral nature of a sport potentially further contributes to differences in pedal asymmetry. 23

CONCLUSION
The double leg squat, in either a bodyweight or weighted condition, represents a foundational movement pattern used in most sport training programs and is highly fundamental to rehabilitation environments. While asymmetries in ground reaction forces tend to be minimized through certain training approaches, the observed functional differences of the recorded kinetic properties in the current study indicate that traditional notions of symmetry may not hold true, even within a population of healthy individuals. While these results add to the existing literature, further work is needed to fully understand the impact of tempo, sport training, and fatigue on the functional outcomes associated with the double leg squat, as it is a frequently-used exercise in rehabilitation and athletic settings. Coaches and practitioners need to consider this preferential use of the right and left legs when developing strength training exercises and sport-specific drills in order to maximize overall task performance for these athletes.


High Dose of Acute Normobaric Hypoxia Does Not Adversely Affect Sprint Interval Training, Cognitive Performance and Heart Rate Variability in Males and Females

Simple Summary Sprint interval training (SIT) is a feasible and time-efficient alternative to classical endurance training that has gained popularity among athletes because of its ability to elicit physiological and cardiorespiratory adaptations in a shorter amount of time than traditional endurance training. Further, popular altitude/hypoxic training techniques include intermittent hypoxic training, in which athletes exercise at submaximal levels under simulated hypoxia while living at sea level (normoxia). Hypoxic exercise is likely a more potent stimulant to upregulate muscle factors (e.g., mitochondrial biogenesis, oxidative, and glycolytic enzymes) than similar normoxic exercise. However, SIT in hypoxia may disturb acute performance indices during sprint intervals. Hypoxia may also impair cognitive function. Acute hypoxia may decrease cognitive performance in areas such as memory and executive functioning. Moreover, males and females may have distinct athletic performance responses to SIT and hypoxia. However, to date, there is no study that has investigated the effects of different doses of acute normobaric hypoxia on SIT and cognitive performance, nor has there been research investigating potential sex-based differences. Abstract Although preliminary studies suggested sex-related differences in physiological responses to hypoxia, the effects of sex on sprint interval training (SIT) performance in different degrees of hypoxia are largely lacking. The aim of this study was to examine the acute effect of different doses of normobaric hypoxia on SIT performance as well as heart rate variability (HRV) and cognitive performance (CP) in amateur-trained team sport players by comparing potential sex differences. In a randomized, double-blind, crossover design, 26 (13 females) amateur team-sport (football, basketball, handball, rugby) players completed acute SIT (6 × 15 s all-out sprints, separated with 2 min active recovery, against a load equivalent to 9% of body weight) on a cycle ergometer, in one of four conditions: (I) normoxia without a mask (FiO2: 20.9%) (CON); (II) normoxia with a mask (FiO2: 20.9%) (NOR); (III) moderate hypoxia (FiO2: 15.4%) with mask (MHYP); and (IV) high hypoxia (FiO2: 13.4%) with mask (HHYP). Peak (PPO) and mean power output (MPO), HRV, heart rate (HR), CP, capillary lactate (BLa), and ratings of perceived exertion (RPE) pre- and post-SIT were compared between CON, NOR, MHYP and HHYP. There were no significant differences found between trials for PPO (p = 0.55), MPO (p = 0.44), RPE (p = 0.39), HR (p = 0.49), HRV (p > 0.05) and CP (response accuracy: p = 0.92; reaction time: p = 0.24). The changes in MP, PP, RPE, HR, CP and HRV were similar between men and women (all p > 0.05). While BLa was similar (p = 0.10) between MHYP and HHYP trials, it was greater compared to CON (p = 0.01) and NOR (p = 0.01), without a sex-effect. In conclusion, compared to normoxia, hypoxia, and wearing a mask, have no effect on SIT acute responses (other than lactate), including PP, MP, RPE, CP, HR, and cardiac autonomic modulation either in men or women.

Introduction
Compared to traditional endurance training, sprint interval training (SIT) is a viable and time-efficient alternative to induce physiological and cardiorespiratory adaptations having become popular among athletes [1]. Indeed, SIT has been reported to improve mitochondrial enzyme activity [2], reduce glycogen utilization and lactate accumulation during work-matched exercise [3], enhance aerobic capacity and performance [4], and improve muscle buffering capacity [5]. Moreover, it is known that hypoxic training can induce upregulation of mitochondrial biogenesis [6,7], oxidative and glycolytic enzymes [6,8,9], and monocarboxylate transporters [10]. Therefore, the application of SIT in altitude/hypoxic environments to enhance endurance and high-intensity exercise performance has recently received unprecedented interest from athletes [11], although it still remains to be explored further.
The SIT in the hypoxia method is of interest in a wide range of team sports as well as endurance sports [12] as it demands remarkably high recruitment of type II fibers [13,14]. Besides its efficacy being dependent on both duration and the sprint:rest ratio, the severity of hypoxia would be also a crucial factor that can affect the aforementioned adaptations. Indeed, Bowtell et al. [15] have reported the highest changes in physiological parameters and performance at F i O 2 : 12% between four different hypoxic conditions (F i O 2 : 12-15%). Further studies have also shown [16,17] that the higher decrease in sprint performance occurred at the higher doses of hypoxia (F i O 2 : 12.5% vs. F i O 2 : 14.5%). However, Kon et al. [18] have reported that there is no difference in capillary blood lactate (BLa) concentration and sprint performance between normoxic (F i O 2 : 21%), and two different degrees of hypoxia conditions (F i O 2 : 16.4% and F i O 2 : 13.6%). Additionally, it has been reported that improvements in VO 2 , time to exhaustion and ratings of perceived exertion (RPE) after a 4-week of SIT were similar between hypoxia (F i O 2 : 13.6%) and normoxia [19], suggesting that the hypoxic degree might be too low to induce more training responses. Therefore, further studies are needed to improve the understanding of the effect of degrees of hypoxia on SIT performance, which may provide positive implications for the programming of the training intervention.
Whilst previous studies have mostly investigated the effect of SIT on metabolic parameters, such as VO 2 , oxidative enzymes, and mitochondrial biogenesis, its impact on heart rate variability (HRV) and cognitive performance (CP) under hypoxic conditions is poorly understood. The HRV is a non-invasive indicator of cardiac activity, with high HRV indicating healthy cardiac activity [20]. The activity of the cardiac autonomic system (CAS) has been reported to decrease at rest in hypoxia [20] compared to normoxia, but both CAS and sympathetic activity seems to be maintained during exercise in hypoxia compared to normoxia [21,22]. Although these data indirectly suggest that hypoxic condition has the potential to influence HRV, empirical evidence to support this, particularly in different degrees of hypoxia, is presently lacking.
Hypoxia can also affect cognitive performance (CP) [23]. Acute hypoxic exposure may reduce CP in domains such as memory and executive functions [24]. Moreover, the use of masks is common during hypoxia simulation; however, the mask may increase breathing difficulty for athletes, creating a psychological strain [25], with a negative psychological effect [26]. However, how CP is affected during SIT under different doses of hypoxia (i.e., high vs. moderate hypoxia) is unknown. Clarification of such a research gap would help to understand the inter-relationships between hypoxia dose and SIT programming variables.
The effects of sex-based differences on SIT and hypoxia are also of interest in a variety of sports, as males and females may manifest different athletic performance responses [27]. Females, compared to males, have been reported to have greater fatigue resistance and better recovery, despite higher cardiovascular strain and RPE in during 8 × 30 m all-Biology 2022, 11, 1463 3 of 12 out sprints [28] and 6 × 4 min high-intensity interval training bouts in normoxia [29]. Females were also found to have lower BLa concentration during hypoxic exercise (F i O 2 : 13%) and higher glucose levels during recovery when compared to males [30]. These observations may be partially based on the finding that sex-dependent differences in intermittent exercise occur during rest intervals since females have been reported to present faster adenosine triphosphate (ATP) recovery [31]. However, if such sex-based differences in SIT performance as well as HRV and CP under different doses of hypoxia (i.e., high vs. moderate) exist remains to be elucidated. Therefore, the aim of this study was to examine the acute effects of different doses of normobaric hypoxia on SIT performance as well as HRV and CP in amateur-trained team-sport players by comparing potential sex differences. Our hypothesis is that hypoxia would affect SIT performance to a greater degree in males compared to females. We also hypothesized that these effects would be greater in a higher dose of hypoxic conditions.

Participants
Thirteen female (age 21 ± 1 years; height 168.0 ± 7.7 cm; body mass 60.7 ± 5.8 kg; VO 2max 50.0 ± 2.1 mL/kg/min; mean ± SD) and thirteen male (age 22 ± 1 years; height 178.2 ± 8.6 cm; body mass 76.3 ± 3.7 kg; VO 2max 54.3 ± 3.4 mL/kg/min) amateur-trained team sport players participated in this study. Participants had, currently or in the previous 3 months, no musculoskeletal injury. Exclusion criteria were pre-existing acute or chronic diseases, having exposure to a real or simulated altitude higher than 1500 m during the previous 3 months., regular smoking, and any travel to altitudes > 2000 m within the 3 months preceding this study. All participants had completed at least 5 h weekly all-out sprint type activities.
An incremental cycling protocol up to exhaustion was performed to determine the VO 2max [32]. The participants were informed about the experimental details, and they gave written informed consent before commencing the study. Participants were informed to refrain from vigorous physical activity, and consumption of caffeine, alcohol and ergogenic aid that might improve performance acutely (i.e., nitrate, l-arginine, l-citrulline and bicarbonate) at least 24 h before each trial. Participants were asked to record their 24-h dietary intake before the first trial and to replicate the same diet before the subsequent trials. The testing sessions were carried out in conformity with ethical standards (i.e., updated version of the Declaration of Helsinki). Sinop University, Human Research Ethics Committee approved the study protocols (decision no: 2021/30).

Study Design
Participants attended the laboratory on five occasions, separated by 72 h. The first visit included the completion of study documentation, baseline anthropomorphic and determination of VO 2max via an incremental cycling protocol up to voluntary exhaustion [32]. Participants also completed a familiarization SIT protocol in which data was collected but was only used to display any learning effects and not for further analyses. Following completion of this initial familiarization visit, participants were assigned to perform the SIT protocol either (I) 900 m (F i O 2 : 20.9%) without wearing a hypoxia generator mask (CON), (II) 900 m (F i O 2 : 20.9%) wearing a hypoxia generator mask (NOR), (III) 2500 m (F i O 2 : 15.4%; MHYP) wearing a hypoxia generator mask, or (IV) 3500 m (F i O 2 : 13.5%; HHYP) wearing a hypoxia generator mask in a randomized, counter-balanced, double-blind, crossover design. A researcher who was not involved in data collection and analyses conducted the arrangements concerning session order and blinding. Participants were not able to see and/or read any data and/or results during exercise. Further, no data and/or results were shared with participants until they completed all experimental sessions. All sessions were conducted during the luteal phase of the female participants' menstrual cycle [33,34] To simulate the specified altitudes during the SIT sessions, the participants wore a mask connected to the Everest Summit II-Altitude Generator (Hypoxico, NY, USA).
To verify hypoxic and normoxic conditions, oxygen was assessed by a pulse oximeter (Hypoxico Oxycon, USA) attached to the participants' fingers during sprint bouts [7,15,17]. The four SIT trials were carried out at the same time of the day (7:00-9:00 a.m.) at the laboratory in which the room temperature (22 ± 1.2 • C) and humidity (55 ± 5.3%) were controlled. HRV was recorded for 5 min before and after (~1 min post) SIT protocol. Following HRV measurements (~45 s post), CP was also measured for 3 min before and after the SIT protocol. HR (lactate scout, USA), RPE and BLa samples were measured at rest, after each sprint and at the end of the test protocol ( Figure 1).
able to see and/or read any data and/or results during exercise. Further, no data and/or results were shared with participants until they completed all experimental sessions. All sessions were conducted during the luteal phase of the female participants' menstrual cycle [33,34] To simulate the specified altitudes during the SIT sessions, the participants wore a mask connected to the Everest Summit II-Altitude Generator (Hypoxico, NY, USA). To verify hypoxic and normoxic conditions, oxygen was assessed by a pulse oximeter (Hypoxico Oxycon, USA) attached to the participants' fingers during sprint bouts [7,15,17]. The four SIT trials were carried out at the same time of the day (7:00-9:00 a.m.) at the laboratory in which the room temperature (22 ± 1.2 °C) and humidity (55 ± 5.3%) were controlled. HRV was recorded for 5 min before and after (~1 min post) SIT protocol. Following HRV measurements (~45 s post), CP was also measured for 3 min before and after the SIT protocol. HR (lactate scout, USA), RPE and BLa samples were measured at rest, after each sprint and at the end of the test protocol ( Figure 1).

SIT Protocol and Computed Performance Indices
The SIT protocol included a 5 min warm-up (cycling at 60 W), six repeated cycling sprints (total time = 11.5 min), and a 3 min cool-down at 60 rpm and 60 W. The SIT trial was conducted in a cycle ergometer (Monark Exercise AB, Vansbro, Sweden), involving 6 × 15 s all-out sprints, separated with 2 min active recovery at 60 W (60 rpm against 1 kg load), against a load equivalent to 9% of body weight. This SIT protocol improved time to exhaustion and VO2max in men and women after 3 weeks [1]. During each sprint, peak (PPO) and mean power output (MPO) were computed and recorded (Monark Anaerobic Test Sofware, Version 3.3.0.0., Vansbro, Sweden), as well as HR, RPE and peripheral oxygen saturation (SpO2) after each sprint.

Heart Rate Variability
Upon arrival at the laboratory and following 15 min of rest in the supine position, HRV was recorded for 5 min before the SIT protocol by using validated equipment (Omega Wave 800, OW, Portland, OR, USA) [33]. HRV was also recorded for 5 min ~1 min after completing the SIT protocol. Three of the seven electrodes used during measurements were thoracic Wilson electrodes and four tarsal limb electrodes. Participants were asked to remain silent and still during the measurements while maintaining their routine respiratory rate. Using validated software (Omega Wave Sport Tech, Portland, OR, USA) automatic records were obtained for the following HRV outcomes: standard deviation of normal-to-normal (NN) intervals (SDNN), standard deviation of successive differences (SDSD), root mean square of successive differences (RMSSD; i.e., parasympathetic activity), total power (TP; variations between NN intervals), the ratio of low-and high-frequency powers (LF/HF; i.e., sympatho-vagal balance), high-frequency power (HF;

SIT Protocol and Computed Performance Indices
The SIT protocol included a 5 min warm-up (cycling at 60 W), six repeated cycling sprints (total time = 11.5 min), and a 3 min cool-down at 60 rpm and 60 W. The SIT trial was conducted in a cycle ergometer (Monark Exercise AB, Vansbro, Sweden), involving 6 × 15 s all-out sprints, separated with 2 min active recovery at 60 W (60 rpm against 1 kg load), against a load equivalent to 9% of body weight. This SIT protocol improved time to exhaustion and VO 2max in men and women after 3 weeks [1]. During each sprint, peak (PPO) and mean power output (MPO) were computed and recorded (Monark Anaerobic Test Sofware, Version 3.3.0.0., Vansbro, Sweden), as well as HR, RPE and peripheral oxygen saturation (SpO 2 ) after each sprint.

Heart Rate Variability
Upon arrival at the laboratory and following 15 min of rest in the supine position, HRV was recorded for 5 min before the SIT protocol by using validated equipment (Omega Wave 800, OW, Portland, OR, USA) [33]. HRV was also recorded for 5 min~1 min after completing the SIT protocol. Three of the seven electrodes used during measurements were thoracic Wilson electrodes and four tarsal limb electrodes. Participants were asked to remain silent and still during the measurements while maintaining their routine respiratory rate. Using validated software (Omega Wave Sport Tech, Portland, OR, USA) automatic records were obtained for the following HRV outcomes: standard deviation of normal-tonormal (NN) intervals (SDNN), standard deviation of successive differences (SDSD), root mean square of successive differences (RMSSD; i.e., parasympathetic activity), total power (TP; variations between NN intervals), the ratio of low-and high-frequency powers (LF/HF; i.e., sympatho-vagal balance), high-frequency power (HF; i.e., vagal activity, low-frequency power (LF; i.e., combination of sympathetic and parasympathetic activity) [35,36].

Cognitive Performance
Mean response accuracy (%) and response times (ms) were collected as CP outcomes, using a modified version of the Flanker task [37,38], with validated software (Inquisit Lab 5.0). Briefly, the participants looked at a white background (with a yellow star at the centre) were five black arrows suddenly appear (in random order, e.g., < < > < < <; > > < > Biology 2022, 11, 1463 5 of 12 >; etc.) during 200 ms. Thereafter, the participants had to respond as quickly and accurately as possible regarding the direction of the middle arrow (i.e., either > or <), by pressing a button with their left or right index finger. Participants had up to 2000 ms from the onset of the stimulus to respond. After 20 practice trials, the participants wore earplugs and performed 100 trials, with an inter-trial interval of 1000 to 2000 ms. The total duration of the test was~3 min.

Pre SIT
Post SIT Pre SIT Post SIT

Discussion
This study examined the acute effects of different doses of normobaric hypoxia on SIT performance, HRV and CP in amateur-trained team-sport players by comparing potential sex differences. The primary findings show that acute normobaric hypoxia with different doses had no effect on SIT performance, HRV and CP between female and male team sports players. However, lower SpO 2 values were observed in HHYP compared to MHYP conditions and females had higher SpO 2 values than males after MHYP and HHYP conditions. Collectively, these findings conflict with our experimental hypothesis and reveal that the acute mild or high dose normobaric hypoxia does not influence SIT performance, HRV and CP in female and male team sports players.
The acute MHYP and HHYP did not provide any difference in PPO and MPO during the SIT protocol compared to normoxia. These observations are consistent with some [15,40], but not all [41][42][43] previous studies. These discrepancy findings between the studies are likely due to the application of considerably different exercise modalities and exercise protocols regarding work-to-rest ratio (e.g., durations of sprint and/or recovery). For example, whilst Brocherie et al. [40] reported a decrease in repeated sprint performance during 6 × 15 s sprints separated with 30 sec recoveries, in hypoxia compared to normoxia, Kon et al. [42] reported no changes in sprint performance during a 4 × 30 s sprint with a 4 min rest. Further, it has been suggested that sprint performance decreases during repeated sprints in hypoxia when the recovery is less than 30 s [40,41]. Together, our findings combined with previous observations reveal that when the recovery periods between sprints are sufficient (2-5 min), the sprint performance will not deteriorate as the energy stores, such as muscle phosphocreatine (PCr), would recover completely although hypoxia can increase non-oxidative glycolysis [40,41]. The present study showed that PPO and MPO were greater in males than females, which is in accordance with others who illustrated that differences between males and females may be attributed to body composition differences [44][45][46]. To the best of our knowledge, this is the first study to focus on sex-based performance differences across SIT under different doses of acute normobaric hypoxia. Previously, only one study assessed the sex-based differences in repeated-sprint performance but performed under only one hypoxic condition (F i O 2 : 13%) [47]. That study reported that sprint performance decreased in hypoxic conditions compared to normoxic conditions, but no differences were found between sexes. Our findings are in line with the study by Smith and Billaut [47] regarding sex-based differences, but inconsistent with regard to sprint performance.
It has been reported that in a hypoxic environment, tissue saturation index and deoxyhemoglobin decrease continuously as the dose of hypoxia increases [48], and physiological changes such as cerebral deoxygenation may have deleterious effects on CP [49]. Surprisingly, our study found no difference in CP between hypoxic conditions. These findings may indicate that the duration of hypoxic exposure was too short due to the low exercise volume and this situation does not provide sufficient physiological stress on the central nervous system. Moreover, our rest interval between sprints (2 min) may have provided adequate time for brain oxygenation, preventing deterioration of cognitive function.
Our findings have shown that HRV variables were not affected by hypoxia. Most studies examining the effect of hypoxia on HRV have been conducted in low-intensity physical activities. In one study, it has been reported that low-frequency component (LF) and LF/high-frequency component (HF) values decrease more after exercise in hypoxia (between 1200-3000 m) compared to normoxia [50]. Another study found that HR recovery decreases after a submaximal workload (5 min) at 2400 m of altitude [51]. Aras and Coskun [52] examined the effect of a single bout of the Wingate test on HRV variables under various levels of altitude (162 m, 1015 m, 2146 m, and 3085 m) in healthy males and females, and reported no changes in HRV variables at any altitudes. However, Botek et al. [53] found significantly decreased vagal activity after exposure to hypoxia at 6200 m in healthy males. The most obvious explanation for the inconsistent findings is because the extreme altitude was used by Botek et al. [53], which is not applicable and practical for at least team-sport athletes. The present study also found no differences between sexes regarding HRV. Those findings are in accordance with previous studies, which showed a lack of differences between sexes related to the cardiac system response to hypoxia [54,55].
We observed higher BLa concentration in HHYP compared to CON and NOR. As the altitude increases, the increase in BLa concentration with the decrease in inhaled oxygen indicates that non-oxidative glycolysis increases [15]. Our findings are consistent with previous studies reporting higher BLa concentration during (I) 3 sets of 6 × 10 s sprints at different doses of hypoxia (FiO 2 : 14.5%, 13.5% and 12.5%) compared to normoxia [17] and (II) during 4 sets of 4 × 4 s sprints in hypoxic condition (FiO 2 : 14%) compared to in normoxic condition [56]. There was a dose-dependent lowering in SpO 2 in the current study as the FiO 2 of the applied gas mixture was decreased. This finding is in line with previous studies reporting greater decreases in SpO 2 during hypoxic exercise [57] and a dose-dependent decrease in muscle oxygenation [58]. This decrease in SpO 2 increases the stress on glycolytic flux and therefore may stimulate the upregulation of this energy pathway (anaerobic) [59].
We acknowledge some potential limitations in our study. Firstly, we did not assess the female menstrual cycle, which might have affected some of our findings. Nonetheless, all testing protocols were completed in 7-8 days, reducing the chances that female participants went through different menstrual phases during this short period of time. Secondly, the assessment of respiratory gases (e.g., oxygen consumption and carbon dioxide production) and ventilation parameters (e.g., tidal volume or minute ventilation) might be helpful parameters to better understand the responses of the cardiac system to different doses of acute hypoxia.

Conclusions
In conclusion, these findings suggest that different doses of acute normobaric hypoxia had no effect on SIT performance, HRV and CP and highlight that there are no sex-based differences in these acute responses in normobaric hypoxia. These observations do not support acute normobaric hypoxia, at least less than 12 min of hypoxia, to alter SIT performance in amateur-trained male and female team-sports players. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to restrictions privacy.


Müller Glia maintain their regenerative potential despite degeneration in the aged zebrafish retina

Abstract Ageing is a significant risk factor for degeneration of the retina. Müller glia cells (MG) are key for neuronal regeneration, so harnessing the regenerative capacity of MG in the retina offers great promise for the treatment of age‐associated blinding conditions. Yet, the impact of ageing on MG regenerative capacity is unclear. Here, we show that the zebrafish retina undergoes telomerase‐independent, age‐related neurodegeneration but that this is insufficient to stimulate MG proliferation and regeneration. Instead, age‐related neurodegeneration is accompanied by MG morphological aberrations and loss of vision. Mechanistically, yes‐associated protein (Yap), part of the Hippo signalling, has been shown to be critical for the regenerative response in the damaged retina, and we show that Yap expression levels decline with ageing. Despite this, morphologically and molecularly altered aged MG retain the capacity to regenerate neurons after acute light damage, therefore, highlighting key differences in the MG response to high‐intensity acute damage versus chronic neuronal loss in the zebrafish retina.

| INTRODUC TI ON
The physiology and structure of the healthy human eye is known to degrade with age. Hallmarks of the ageing human retina include tissue thinning, neuronal loss, especially in the macula, and reduced visual function (Arden & Jacobson, 1978;Marshall, 1987;Salvi et al., 2006;Skalka, 1980;Slataper, 1950;Weale, 1975). Accordingly, ageing is a significant risk factor for degeneration and disease in the retina, such as age-related macular degeneration or primary open angle glaucoma (Al-Ubaidi et al., 2013;Marshall, 1987). Retinal degeneration is often a process that plays out over months or years, whereby neurons gradually die leading to dysfunction (Marshall, 1987;Salvi et al., 2006). Regenerative-based therapies, such as stimulating endogenous Müller Glia (MG) to regenerate dying neurons, offer a great promise for the treatment of various types of degenerative blinding conditions, including those associated with ageing.
Thus, determining the effects of ageing on the regenerative potential of the retina is an important consideration for the efficacy of such potential treatments going forward.
Müller Glia are the principal cells tasked with supporting and maintaining the retina throughout life. They provide retinal neurons with a myriad of supportive functions, including trophic support, neurotransmitter recycling and energy metabolism (Reichenbach & Bringmann, 2013). While these functions are critical for healthy retinal function, MG also have a prominent role during disease and after neuronal insult. In the mammalian retina, neuronal damage results in a MG gliotic response, involving the up-regulation of stress proteins, proliferation and morphology changes (e.g. hypertrophy) (Bringmann & Wiedemann, 2012). This response is thought to be neuroprotective initially, but may ultimately culminate in dysfunction and death via loss of metabolic support or tissue integrity (Bringmann et al., 2009). While the mammalian retina lacks significant regenerative capacity (Jadhav et al., 2009;Ooto et al., 2004), it has been shown that re-introduction of key molecular signals into MG can stimulate the genesis of new neurons in mice after acute damage (Jorstad et al., 2017(Jorstad et al., , 2020Karl et al., 2008), raising hopes that identifying the molecular mechanisms driving regeneration may provide therapeutic targets that we can exploit in the future. One such example is Hippo/Yap signalling, which has been identified as a signalling pathway that is altered after neuron degeneration and injury within MG cells (Hamon et al., 2017(Hamon et al., , 2019Rueda et al., 2019).
Accordingly, intensive efforts have been made to uncover the molecular mechanisms regulating these endogenous regenerative responses in the retinas of "lower vertebrates", such as fish and amphibians, where MG cells have the capacity to regenerate the retina after neuronal damage (Goldman, 2014). However, even though retinal regeneration is largely studied in these models, such as in the zebrafish (Fausett & Goldman, 2006;Fischer et al., 2002;Lenkowski & Raymond, 2014;Raymond et al., 2006;Thummel et al., 2008;Wan & Goldman, 2016), mechanisms are generally investigated in young adults, and regeneration is stimulated by inducing a high-intensity acute damage injury, which is likely to differ from the slowly accumulating chronic damage associated with natural ageing. In the zebrafish retina, after high-intensity acute damage, such as intense light or toxins (Lenkowski & Raymond, 2014;Thomas et al., 2012), MG undergo a brief reactive gliosis-like phase that transitions into a regenerative response: a de-differentiation and proliferation of the MG progenitor cell to specifically generate new neurons and restore vision (Eastlake et al., 2017;Thomas et al., 2016;Thummel et al., 2008). In contrast to high-intensity acute injury, damage to the retina caused by degenerative disease in ageing will manifest itself over much longer timescales (wildtype zebrafish can live up to c.43 months in the lab (Carneiro et al., 2016)) and results in the gradual accumulation of DNA damage and cell death (Carneiro et al., 2016;Henriques et al., 2013;Salvi et al., 2006). As such, chronic degeneration in ageing may significantly differ from injury models, especially in its capacity to induce a regenerative response from glia.
While multiple acute injuries do not appear to reduce the overall regenerative response of MG (Thomas et al., 2012), the effects of natural chronic age-related damage on the MG regenerative potential has not been determined.
The zebrafish offers a suitable model for studying regeneration in the context of ageing, as it displays key human-like hallmarks of ageing such as shorter telomeres and associated DNA damage (Carneiro et al., 2016). Accordingly, the well-established tert −/− mutant zebrafish have impaired regeneration, both in steady-state in tissues such as the gut (Anchelin et al., 2013;Henriques et al., 2013) and in response to injury, in the heart (Bednarek et al., 2015).
The tert −/− model therefore offers the possibility of identifying telomerase-dependent and telomerase-independent molecular drivers of regeneration in a context of accelerated ageing. Moreover, the use of premature ageing models have provided key molecular insights into the mechanisms driving the ageing process and can contribute to the identification of therapeutic targets for age-associated dysfunction and disease (Kubben & Misteli, 2017).
In this study, we used the naturally aged wildtype (WT) zebrafish and the prematurely aged tert −/− as models to study the impact of ageing and telomerase function on the maintenance of neuronal structure and regenerative capacity of MG cells. We started by confirming that naturally aged retinas display known hallmarks of retinal ageing, including tissue thinning, accumulation of DNA damage and neuronal loss (Van Houcke et al., 2019), which we show are accompanied by significant histopathological abnormalities in the retina. However, we now show that most of this damage is not accelerated in the absence of telomerase (tert −/− mutant), suggesting these to be largely telomerase-independent.

K E Y W O R D S
ageing, degeneration, Müller glia, proliferation, regeneration, retina, telomerase, Zebrafish Interestingly, the accumulation of chronic damage with ageing is not sufficient to stimulate MG to proliferate or replace lost neurons. Instead, ageing leads to morphological and molecular aberrations in MG and loss of vision, recapitulating hallmarks of human retinal degeneration with advancing age. Moreover, we show that MG cells express Yap throughout lifespan, a key molecule involved in the regenerative response (Hamon et al., 2019;Rueda et al., 2019), but that this expression decreases with advancing age. Despite reduced Yap levels, aged MG cells are still capable of regenerating neurons in old zebrafish, after acute light injury.
We, therefore, identify differences in the MG response to highintensity acute damage versus chronic damage, an important consideration for potential therapies aiming to stimulate endogenous regenerative mechanisms to treat human retinal disease.

| The aged zebrafish retina displays neurodegeneration independently of telomerase
Similarly to humans, the zebrafish retina consists of three nuclear layers separated by two synaptic plexiform layers (Easter & Nicola, 1996). The nuclear layers consist in the outer nuclear layer (ONL), containing photoreceptors; the inner nuclear layer (INL) containing bipolar cells (BCs), amacrine cells (ACs), horizontal cells (HCs) and MG; and the ganglion cell layer (GCL) mainly containing retinal ganglion cells (RGCs) (see diagram in Figure 1a). Further, RGCs, ACs and BCs are neurons that come together to make connections in the major synaptic neuropil, called the inner plexiform layer (IPL; Figure 1a). The zebrafish central retina is considered fully specified by 73 h post-fertilisation (hpf) (Easter & Nicola, 1996) and continually grows at the periphery throughout life due to proliferation of the ciliary marginal zone (CMZ) (Easter & Nicola, 1996). Thus, the central region of the adult retina can be considered the "oldest", as it is largely generated during early developmental stages, and where there is very little proliferation to generate new neurons (Fu et al., 2018;Julian et al., 1998), with the exception of rods (Johns & Fernald, 1981;Julian et al., 1998;Nelson et al., 2008;Otteson et al., 2001). In contrast, the peripheral region of the retina is continually expanding at the margins due with the newest born cells being found adjacent to the CMZ It has recently been shown that the ageing WT zebrafish retina shows degeneration phenotypes with advancing age (Fu et al., 2018;Van Houcke et al., 2019). Telomerase is known to be important for proliferation and regeneration in the zebrafish (Anchelin et al., 2013;Bednarek et al., 2015;Carneiro et al., 2016;Henriques et al., 2013); however, apart from its described role in the human retinal pigment epithelium (RPE) (Drigeard Desgarnier et al., 2016), its specific role in the ageing retina remains unclear. Here, we characterised key known hallmarks of human retina ageing (Eriksson & Alm, 2009), including retina thinning, morphology and histopathological alterations. We show that the WT and tert −/− zebrafish central retina progressively thins with ageing ( Figure 1b, b',c,c',c''), consistent with previous studies (Eastlake et al., 2017;Thomas et al., 2016). This retinal thinning is specific to the central retina, as the peripheral retina maintains its thickness over-time (Figure 1c',c'' and Figure S1). Thinning of the central retina is accompanied by several morphological and histopathological alterations, including cellular spatial rearrangement, cellular atrophy and the development of vacuoles in the RPE (Figure 1c-c'' and Figure S2). However, apart from specific alterations in the RPE, we show that this occurs largely independently of telomerase, as the telomerase-deficient model tert −/− displays retinal thinning and most morphological and histopathological alterations at the same rate as the WT (Figure 1b',c').
To determine which type of neuronal loss was underpinning retinal thinning, we used immunohistochemistry with several molecular markers specific for different neuronal populations from young to old ages, in the presence and absence of telomerase (tert −/− ). As  (Lo, 2019) and we show that long double-cones in the zebrafish retina undergo structural changes (short and/or disorganised) with ageing, shown by ID4 staining (Figure 2h,h'). To confirm the photoreceptor outer segment disorganisation, we stained rod and cone outer segments with the zpr3 antibody ( Figure S3a) and red/green double cones with the zpr1 antibody ( Figure S3b).
Zpr3 staining clearly shows the shortened and misaligned outer segments in the WT aged retina, but is not accelerated in tert −/− ( Figure   S3a (Fu et al., 2018). These defects are also not accelerated in the tert −/− at the ages tested, suggesting these are likely telomerase-independent phenomena. | 5 of 20 MARTINS eT Al.

| MG do not proliferate in response to chronic retinal neurodegeneration with ageing
It is well-established that MG in the zebrafish retina proliferate and regenerate lost neurons after high-intensity acute damage (Jadhav et al., 2009;Jorstad et al., 2017;Ooto et al., 2004). Although the progressive age-related retinal neurodegeneration would suggest a lack of regenerative response by MG, it remained unclear whether this degeneration was being counteracted, at least to some degree, by proliferation in the aged retina. To test this, we used a 5-ethynyl-2′-deoxyuridine (EdU) pulse-chase strategy to identify any cell divisions in the retina, from young to old ages, which would be indicative of potential regeneration. To characterise the steadystate regenerative capacity of the central and peripheral CMZ until old age, we carried out a 3-day pulse of EdU followed by 0-and 30- ing that there is no compensatory proliferation from the CMZ in response to neuronal loss with ageing. Finally, the fact that there are very low and decreasing levels of proliferation in wildtype ageing could explain why removing telomerase (tert −/− ) has no further significant effect. As proliferation of MG is the primary source of neurons after acute injury in fish (Bernardos et al., 2007;Fausett & Goldman, 2006), we wanted to determine whether there could be any small compensatory proliferation over-time, within MG cells specifically.
To do so, we co-labelled cells with the MG specific marker glutamine synthetase (GS) and counted the GS-positive; EdU-positive cells. what has been reported to occur in response to high-intensity acute damage (Jadhav et al., 2009;Jorstad et al., 2017;Ooto et al., 2004), our data show that age-related chronic cell death does not trigger proliferation of MG cells. Finally, rods can originate from rod-specific progenitors into adulthood (Johns & Fernald, 1981;Julian et al., 1998), which are found in the ONL and are derived from MG that slowly divide in the WT retina (Bernardos et al., 2007;Nelson et al., 2008;Otteson et al., 2001;Raymond et al., 2006). To test whether there is any compensatory proliferation with ageing occurring specifically from rod-specific progenitors found scattered throughout the ONL of the retina (Johns & Fernald, 1981;Stenkamp, 2015), we

| Zebrafish vision declines with ageing, independently of telomerase
Loss of visual acuity and contrast sensitivity with advancing age in humans has been well documented (Marshall, 1987;Salvi et al., 2006). Here, we report that the presence of retinal neuron loss and tissue thinning in the aged retina is largely independently of telomerase (tert). Therefore, to determine whether the molecular and structural changes we observe have a pathological consequence on the retinal function, and whether this is accelerated in the absence of telomerase, we tested visual acuity in WT and tert −/− zebrafish, from young to old ages. Visual testing in the zebrafish has been used to screen for mutants with defects in retinal development and function (Brockerhoff et al., 1995;Gross et al., 2005;Lessieur et al., 2019;Neuhauss et al., 1999), but were yet to be tested in the context of ageing. The optokinetic response (OKR) is used to measure innate visual responses and provides readout of visual acuity (Brockerhoff et al., 1995;Tappeiner et al., 2012)  unlike what is described in other tissues (Carneiro et al., 2016). We, therefore, focused on characterising the morphological and molecular changes underpinning the lack of MG regenerative responses in the ageing WT retina from here onwards.

| MG become morphologically altered with ageing
MG respond to damage or injury in most, if not all, retinal degenerative diseases (Bringmann et al., 2009). In the mammalian retina alterations in MG morphology is a hallmark of MG response after retinal damage (Bringmann & Wiedemann, 2012), but also commonly observed in the aged retina (Telegina et al., 2018). The characteristic mammalian response is gliosis, whereby MG morphology becomes aberrant, accompanied by changes in expression and localisation of structural proteins like glial fibrillary acidic protein (Gfap) (Bringmann et al., 2009;Reichenbach & Bringmann, 2013). As we do not observe a MG regenerative response to neurodegeneration, but we do observe vision loss in naturally aged zebrafish, we wondered whether zebrafish MG were undergoing an ageing-associated damage response similar to mammals. Here, we labelled MG using the well-described GS antibody and characterised MG morphology and organisation using the markers Gfap, Vimentin and cellular retinaldehyde-binding protein (Cralbp) (Garcia & Vecino, 2003;Verardo et al., 2008). Although the number of MG cells is maintained in the central retina throughout the zebrafish lifespan ( Figure S5a

| Müller glia express reduced levels of Yap in old age
As we observe morphological alterations in MG cells and a lack of MG proliferation in response to age-associated chronic damage, we next asked whether aged MG have altered molecular expression of Yap, a key molecule in the retinal regenerative process (Hamon et al., 2019). Specifically, when Hippo signalling is activated, LATS1/2 kinases phosphorylate the transcriptional co-factor Yap, leading to its degradation. Importantly, Yap expression is required for MG regeneration (Hamon et al., 2019) and its expression goes up in reactive MG cells after acute damage (Rueda et al., 2019). We therefore tested whether Yap expression was affected in ageing and in MG specifically. Here we show, by immunohistochemistry, that Yap is expressed in MG until old age (Figure 5a-c), where it is thought to be required for the maintenance of MG proliferative capacity (Rueda et al., 2019). Nevertheless, overall expression levels in the eye are reduced, as assessed by western blot (Figure 5d,d', and Figure S6).
As such, MG retain the expression of a critical molecular player required for the regenerative process after damage, but its levels are significantly reduced. Given this, it was still unclear whether the F I G U R E 2 Zebrafish retina undergoes neurodegeneration with ageing, independently of telomerase. The central retina immunolabelled with HuC/D and PKC (amacrine in magenta and bipolar cells in green, respectively), in both WT and tert −/− , in (a) young (5 months)  naturally aged fish could regenerate their retina in response to a high-intensity acute injury.

| Müller glia retain the ability to regenerate after acute damage until old age
Studies on retinal regeneration typically focus on the young adult retina and have not taken old age into account for the regenerative response. There is evidence from ex vivo mouse explants that MG neurogenic competence in response to neuronal death is limited with advancing age (Loffler et al., 2015); however, this study was restricted to early post-embryonic stages and not more mature adult stages. Several high-intensity acute damage paradigms induce retinal neuron death and elicit a regenerative response in zebrafish (Bernardos et al., 2007;Cameron, 2000;Hanovice et al., 2019;Ranski et al., 2018). Repeated damage to the adult retina, specifically between 9-18 months of age, which is still considered young for a WT fish, results in MG continuously re-entering the cell cycle to proliferate and regenerate lost neurons, although they do show signs of chronic activation after repeated insults (Ranski et al., 2018). However, it remains unclear whether MG retain their ability to proliferate and regenerate the retina throughout life, and whether altered MG, such as those often occurring in retinal disease or old age retain proliferative capacity to replace damaged neurons. Since we show that MG retain Yap expression into old ages, albeit at reduced levels, it remained possible that aged zebrafish retinas could still regenerate in response to high-intensity acute damage. To test this, we used the light-damage model where aged albino zebrafish, at three stages of their lifespan, were treated with light to elicit photoreceptor damage. To detect increased proliferation of MG in response to damage, a 3-day pulse of bromodeoxyuridine (BrdU) was performed, followed by a 28-day chase period (Figure 6a Figure S7). Together, these results suggest that the regenerative response remains intact throughout zebrafish lifespan and the molecular and morphological alterations in aged MG does not impact their regenerative capacity in old age.

| DISCUSS ION
Using a combination of cell labelling strategies throughout adulthood into old age, we show that the zebrafish retina retains its potential to regenerate in response to acute damage into old age. Therefore, the lack of compensatory proliferation in response to chronic, ageassociated cell death in the ageing zebrafish retina is not due to a loss of capacity of MG to proliferate per se, but likely due to the absence or insufficient levels of the required stimuli for MG proliferation and regeneration.

| Telomerase-dependent and telomeraseindependent hallmarks of zebrafish retinal ageing
Previous work has suggested that telomerase and telomere length are important for human retinal health. In particular, the RPE has been reported to have shorter telomeres than the neural retina, and it accumulates senescence over time (Drigeard Desgarnier et al., 2016). Accordingly, reactivation of telomerase has been described to ameliorate symptoms of age-associated macular disease (Dow & Harley, 2016;Rowe-Rendleman & Glickman, 2004) and telomerase activators are currently in clinical trials (e.g. NCT02530255).
However, retinal homeostasis requires more than RPE maintenance.
It requires a steady-state level of proliferation of different cell types F I G U R E 3 Aged zebrafish retina does not show signs of regeneration in response to spontaneous cell death and neuronal loss. (a) Schematic figure of the experimental design: 3-day pulse of EdU, by IP injection, followed by 0-or 30-day chase. We anticipate that in a healthy young fish the retina has some cells proliferating in the CMZ, which over-time will replace older cells in the central retina. In the case of injury, we anticipate that there will be elevated levels of proliferation in peripheral and central retina to replace the dead cells. involved in multiple aspects of retina function. As in other proliferative tissues (Anchelin et al., 2013;Carneiro et al., 2016;Henriques et al., 2013), it would be expected that telomerase levels would influence zebrafish retina homeostasis. We, therefore, hypothesised that the zebrafish retina would degenerate in a telomerase-dependent manner, leading to vision loss. However, our data suggest that, apart from aberrations in the RPE, most age-related changes described here in the naturally aged zebrafish retina, including vision loss, are largely telomerase-independent, since depletion of telomerase (tert −/− ) does not accelerate or exacerbate any of these phenotypes.
Telomere dysfunction is known to affect mostly highly proliferative tissues (reviewed in Henriques and Ferreira, (2012)) and what our data show is that, in the region most affected by ageing phenotypes, the central retina, there is very little proliferation to start with, potentially explaining why telomerase depletion has no further significant effect.

| Chronic vs. high-intensity acute damage in MG responses: a "tipping point" required to stimulate regeneration in ageing?
Regeneration studies so far have relied on several damage paradigms, including phototoxic (Ranski et al., 2018) and ouabaininduced lesions (Mitchell et al., 2019), which induce rapid cell death post-insult. While high-intensity acute damage models are suitable to explore the cellular and molecular mechanisms underpinning the regenerative potential of the retina, they do not test whether this regenerative response is also occurring with natural ageing in the retina. Our results show that zebrafish develop vision loss with ageing and that this is accompanied by retinal neurodegeneration and MG morphological and molecular aberrations, rather than proliferation and replacement of dying neurons. This is counterintuitive to what would be expected based on the well-known regenerative response to neuronal death by MG. Nevertheless, we show that aged zebrafish retinas maintain their regenerative response and capacity after acute injury. This study, therefore, shows that there are critical differences between the MG regenerative response to the neuronal loss induced by chronic damage in ageing and after high-intensity acute injury.
"Natural ageing" and associated neuronal loss play out over months or even years and we now show that this does not elicit the same regenerative response as high-intensity acute damage. This may be because the slow degeneration is not producing a strong enough signal to induce MG to undergo the cellular and molecular processes of regeneration. It has been shown that the level of cell death can induce a differential response of MG cells. Whilst a large amount of rod death causes a regenerative response, small amounts do not (Lessieur et al., 2019;Montgomery et al., 2010;Thomas et al., 2012). Furthermore, there may be distinct differences in signals released after apoptosis or necrosis in ageing vs damage models (Bergmann & Steller, 2010;Nelson et al., 2013). In support of this concept, recent work suggests that there may be key signalling differences underpinning the difference between a "regenerative" or a "reparative" response to injury (Conedera et al., 2020). Thus, we propose that a molecular signal, or expression changes of such signal, will regulate the "tipping point" required to elicit a MG regenerative response in ageing.
Research in the context of acute damage paradigms in the zebrafish retina has uncovered many of the molecular mechanisms regulating this regenerative response (reviewed in Goldman, 2014;Julian et al., 1998). For instance, proliferation appears to be a key mechanism for the initiation of the regenerative response as blocking proliferation after damage in the zebrafish retina results in MG alterations, and not regeneration (Thummel et al., 2008), similarly to humans.
Moreover, age-associated degeneration of retinal neurons and their synapses, as we observed to occur in this study, may result in a loss of neurotransmitter release, such as gamma-aminobutyric acid (GABA), which has been shown to facilitate the initiation of MG proliferation (Rao et al., 2017). Thus, dysregulation of neurotransmitters upon neurodegeneration could inhibit the key molecular pathways regulating regeneration. Alternatively, the initial inflammatory response has also been shown to be determinant for the repair process. Upon light-induced retinal damage, overexpression and subsequent release of tumour necrosis factor (TNF) by apoptotic photoreceptors seems to induce MG proliferation (Iribarne et al., 2019;Thomas et al., 2016).
After high-intensity acute damage, there is also an increased number of microglia in the retina (Mitchell et al., 2018(Mitchell et al., , 2019 and they have been shown to be essential for retinal regeneration (Conedera et al., 2019;White et al., 2017). In contrast, we show that microglia numbers do not increase with ageing in the zebrafish retina, and therefore, may compromise the regenerative response. Nonetheless, it is important to consider that the available immunohistochemistry techniques to identify microglia numbers have a few limitations. Namely, there may be a spike in microglia number in the aged retina that is rapidly resolved and missed at our time points or microglia may undergo F I G U R E 4 Zebrafish vision declines with ageing, which is accompanied by signs of glial morphological alterations. (a) OKR assay was performed by immobilising the fish in between soft sponges, inside a petri dish containing water, placed in the centre of a rotation chamber. The walls of the rotation chamber had 0.8 mm-thick black and white stripes and the chamber was maintained at a constant velocity of 12 rpm throughout the experiment. signal, and the level required for inducing the response, will be critical to stimulate regeneration in the human retina. It is very promising that mammalian MG can be molecularly manipulated to increase their regenerative potential after damage (Jorstad et al., 2017). However, for these therapies to be truly transformative for human blinding conditions this regenerative capacity must be paired with the appropriate "damage" stimuli since using acute light damage or toxins in humans is unlikely to be therapeutically feasible.

| MG response to chronic damage in ageing
The regenerative capacity of vertebrate tissues tends to decrease after repeated injury and as animals advance in age (Ranski et al., 2018). In the context of the central nervous system, this is aggravated by gliosis, a reactive change in glial cells in response to damage. In the mammalian retina, MG cells undergo gliosis in response to damage and in many retinal degenerative diseases (Bringmann et al., 2009). However, in the zebrafish retina MG cells undergo the initial reactive gliotic response after acute damage but quickly shift to the regenerative pathway. Here, we show that while we observe morphological and molecular alterations in MG cells in response to agerelated neurodegeneration, MG in the aged retina retain their ability to regenerate in response to high-intensity acute damage of photoreceptors. This provides evidence that regenerative mechanisms in tissues that have been damaged or are degenerating remains possible. Importantly, the fact that MG are still capable of proliferating in response to a high-intensity acute injury in old animals, shows that the lack of regeneration in the context of ageing is not due to an intrinsic inability of MG to proliferate per se, suggesting that it is the stimuli itself that is different/insufficient in the context of ageing. Mechanistically, in contrast to what has been described in high-intensity acute damage models, where Yap levels go up (Rueda et al., 2019), we show that overall Yap levels decrease in the chronic damage setting of ageing. It is, therefore, a likely possibility that at least one of the key stimuli missing may be one that is responsible for switching off Hippo signalling pathway, preventing Yap degradation.

| CON CLUS IONS
Our work demonstrates that, in the context of age-induced neuronal degeneration, zebrafish MG undergo degeneration, rather than regeneration. This resembles what occurs in the aged human retina as well as in many human retinal degenerative diseases. Importantly,
The telomerase mutant line tert AB/hu3430 was generated by Nethyl-nitrosourea mutagenesis (Utrecht University, Netherlands; Wienholds, 2004). It has a T→A point mutation in the tert gene and is available at the ZFIN repository, ZFIN ID: ZDB-GENO-100412-50, from ZIRC. The fish used in this study are direct descendants of the ones used previously (Carneiro et al., 2016;Henriques et al., 2013), by which point it had been subsequently outcrossed five times with WT AB for clearing of potential background mutations derived from the random ENU mutagenesis from which this line was originated.
The tert hu3430/hu3430 homozygous mutant is referred to in the paper as tert −/− and was obtained by incrossing our tert AB/hu3430 strain.
In order to study age-related phenotypes in the zebrafish retina, in this study, we use an age >30 months old fish for what we consider old in WT (in the last 25-30% of their lifespan), and we consider the tert −/− old fish at the equivalent age (>12 months) that corresponds to the last 25-30% of their lifespan, approximately. In specific, "Old" was defined as the age at which the majority of the fish present age-associated phenotypes, such as cachexia, loss of body mass and curvature of the spine. These phenotypes develop close to the time of death and are observed at >30 months of age in WT and at >12 months in tert −/− (Carneiro et al., 2016;Henriques et al., 2013).
In addition, we used adult albino zebrafish for retinal regeneration studies (described in detail below). Importantly, none of the animals included in this study displayed visible morphological alterations in the eyes (e.g. cataracts).

| Optokinetic response (OKR) assay
Fish were anaesthetised in 4% tricaine methanesulfonate (MS-222; Covetrus, pharmaceutical grade) and placed in a small bed-like structure made of sponge, located inside a small petri dish containing fish water. During the experiment, fish were maintained still by the strategic use of needles that sustained the sponges close to the fish so that the fish could not move. The petri dish was then placed inside a rotation chamber with black and white striped walls (8 mm-thick stripes). After fish recovered from anaesthesia, the trial began, and the walls of the chamber started rotating at 12 rotations per min (rpm; for 1 min to the left side followed by 1 min to the right side).
Eye movements were recorded using a digital camera throughout the experiment. After the experiment, the number of eye rotations per minute was measured by video observation and manually counting. The counting was performed blindly by two independent researchers. In the end, the results were normalised for the WT young from the same day/batch, in order to control for different days of experiments.

| Intense light-damage paradigm with BrdU incorporation
A photolytic damage model in adult albino zebrafish was utilised to destroy rod and cone photoreceptors and elicit a regenerative response (Thomas et al., 2012). Briefly, adult albino zebrafish were dark-adapted for 10 days prior to a 30 min exposure to ~100,000 lux from a broadband light source. Next, fish were exposed to ~10,000 lux of light from four, 250 W halogen lamps for 24 h.
Following 24 h of light treatment, fish were transferred to a 1 L solution containing 0.66 g of NaCl, 0.1 g Neutral Regulator (SeaChem Laboratories, Inc.), and 1.5 g BrdU (5mM; B5002; Sigma-Aldrich) for 48 h. This timeframe for BrdU incubation was based on previous studies in order to label MG cell-cycle re-entry (Garcia & Vecino, 2003). Following a 48-h incubation in BrdU, the fish were split into two groups: one group was euthanised by an overdose
Briefly, fish were injected with 5 μl of 10 mM EdU diluted in dimethyl sulfoxide (DSMO), by intraperitoneal (IP) injection, for 3 consecutive days (3-day pulse). In order to differentiate proliferating cells and low-proliferative EdU-retaining cells, the fish were separated into two groups: 0-day chase and 30-day chase groups. The fish from the first group were culled 2 h 30 min after the last injection of Edu, whereas the fish from the second group were culled 30 days after the last injection of EdU. After culling, whole fish were processed for paraffin-embedded sections as described above. In order to detect EdU labelling in paraffin-embedded sections, the slides were deparaffinised, hydrated, underwent antigen retrieval, were The incubation in the secondary antibody was performed as previously described.

| Imaging and quantifications
Paraffin-embedded sections were imaged by epifluorescence microscopy, using a DeltaVision microscope with a 40× oil objective.
Cryosections were imaged by laser scanning confocal imaging, using a Leica SP5 microscope, Nikon A1 Confocal microscope or a Zeiss 900 Airyscan 2 confocal, with a 40× oil objective. In either paraffin or cryosections, multiple 0.2-0.6 μm thick z-stacks were acquired in order to capture the whole retina region. For each staining, a total of 4 images were taken per retina, 2 from central and 2 from peripheral retinal. The central retina was defined to be the centre point between opposing CMZs (a minimum of ~1000 μm from the periphery).
The peripheral retina was defined as the tissue directly adjacent to the CMZ.
In order to quantify the alteration in the staining patterns, a zprojection was generated using ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, https://imagej.nih.gov/ij /, 1997-2018.) and three boxes of 100 × 100 μm were drawn in each field of view.
Retinal thickness was measured in DAPI stained paraffin sections, using the software ImageJ to measure the individual layers and overall layers thickness as indicated in the diagram in Figure 1a. For key protein staining, the total number of positive cells was manually counted for each label.
Ribeye A, 1D4, ZO1, ZPR3 and Gfap staining were an exception to this, for which a qualitative assessment was performed instead.
To do so, structural and morphological defects were identified as follows. For the outer segments of long double-cones (1D4) staining, as young WT retinas usually display long and organised outer red green cones, and these were counted manually and their length was measured using image J ( Figure S3).
Finally, raw images were used for quantification purposes. The images were then processed with Adobe Illustrator 21.0.2 for display purposes.

| Protein extraction, quantification and Western blotting
Adult fish were culled by overdose of MS-222, followed by confirmation of death. Dissected eyeballs were snap-frozen in liquid nitrogen and kept at −80°C until protein extraction, as follows.
Frozen tissue was homogenised in 100 μl of radioimmunoprecipi- Immunoblotting was performed as follows. Membranes were blocked using Intercept ® TRIS buffered saline (TBS) Blocking Buffer for 1 h at RT and incubated with target primary antibody (see Table 1 for details) in TBS Blocking Buffer 0.1% Tween-20 (Sigma-Aldrich, Missouri) ON at 4°C, with mild shaking. Secondary antibody detection was performed after washing the primary antibody with 1X TBS (50 mM Tris-Cl, pH 7.5, 150 mM NaCl (Sigma-Aldrich)) with 0.1% Tween-20. Primary antibody detection was performed using

| Statistical analysis
Statistics were performed using the GraphPad Prism v7.00. Normality was assessed by the Shapiro-Wilk test. For normally distributed data where most of the groups had a sample size of ≥5, unpaired t-test was used to compare 2 data points and one-way ANOVA followed by Bonferroni post hoc test was used to compare more than 2 data points. For non-normally distributed data and/or data containing less than 5 animals in most of the groups Mann-Whitney test and Kruskal-Wallis tests were used instead. Two-way ANOVA followed by Tukey's multiple comparisons was used in order to compare more than 2 data points in 2 different groups (genotypes). Chi-square was performed to analyse structure and morphological changes in the retina based on qualitative assessment, having taken into account the number of animals per group displaying defects versus not displaying defects. A critical value for significance of p < 0.05 was used throughout the analysis. There were no repeated measurements performed in this study.

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets and source data generated during and/or analysed during the current study are available from the corresponding author upon reasonable request.